Project Summary The molecular targets for vitreoretinopathy and uveitis (intraocular inflammation) are poorly understood. This proposal builds on our exciting discovery of a calcium-activated protease, calpain-5 (CAPN5), as the cause of Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy (ADNIV), and provides insight into structure and function of CAPN5. CAPN5 is the first nonsyndromic uveitis gene, and this discovery provides an unprecedented starting point for investigating the substantial gap in our understanding of the molecular basis of ocular inflammation. Until recently, the CAPN5 structure had been undetermined, hindering the investigation of its mechanism of action. Since our last submission, we solved the 3.0 crystal structure of the wild-type CAPN5 protease core. Structural studies of CAPN5 ADNIV disease mutants will inform how these mutations alter CAPN5 structure and function. Our long-term goal is to identify new therapeutic targets for uveitis. Our objective is to use x-ray crystallography and other biochemical techniques to study the structural mechanisms of CAPN5 activation. Our central hypothesis is that ADNIV mutants cause the CAPN5 protease core to adopt a closed conformation, stabilizing the active site, and relieving the enzyme from calcium dependence. This overactive CAPN5 then aberrantly cleaves proteolytic targets, contributing to the ADNIV disease phenotype. Insight gained from this study may eventually be used to design therapies for ADNIV and other calpain-associated pathologies. My studies will test the hypothesis through three specific aims: (1) Identify natural CAPN5 peptide substrates and determine their specificity, (2) Use x-ray crystallography to define the structure of CAPN5 mutants, and (3) Determine the function of extensible loops in CAPN5 activity. Impact. We expect to gain not only a better understanding of calpain activity in eye disease, but also to identify new lead compounds to design inhibitors against CAPN5.